The role of patch testing in the management of oral lichenoid reactions.

BACKGROUND AND OBJECTIVES The distinction of oral lichenoid reactions from oral lichen planus may be difficult in a clinical setting. Our aims were to ascertain the utility of patch testing to confirm the association of oral lichenoid reactions with dental restorations and to identify the benefits of replacement of restorations, primarily made of amalgam. METHODS Patients seen in an oral medicine unit over a 10-year period diagnosed with oral lichenoid reactions, with oral lichen planus resistant to treatment or with atypical lichenoid features were included in this study. All had been subjected to skin patch testing. Histopathology reports blinded to patch test results were scrutinized. Patch-test-positive subjects were advised to have their restorations replaced. All were followed up to determine disease resolution for at least 3 months thereafter. RESULTS Among 115 patients, 67.8% patients reacted positive to a dental material and nearly a quarter to mercury or amalgam. No correlation was found between pathology and skin patch testing results (P = 0.44). A total of 87 patients were followed up in clinic, and among 26 patch-test-positive patients who had their amalgam fillings replaced, moderate to complete resolution was noted in 81%. CONCLUSIONS Skin patch testing is a valuable tool to confirm clinically suspected oral lichenoid reactions. Pathology diagnoses of oral lichenoid reactions did not correlate with patch test results. Prospective studies are needed to ascertain that a clinically suspected oral lichenoid reaction with a positive patch test result may resolve after the replacement of amalgam fillings.

Modeling the Effects of Size on Patch Dynamics of an Inert Tracer

Mesoscale iron enrichment experiments have revealed that additional iron affects the phytoplankton productivity and carbon cycle. However, the role of initial size of fertilized patch in determining the patch evolution is poorly quantified due to the limited observational capability and complex of physical processes. Using a three-dimensional ocean circulation model, we simulated different sizes of inert tracer patches that were only regulated by physical circulation and diffusion. Model results showed that during the first few days since release of inert tracer, the calculated dilution rate was found to be a linear function with time, which was sensitive to the initial patch size with steeper slope for smaller size patch. After the initial phase of rapid decay, the relationship between dilution rate and time became an exponential function, which was also size dependent. Therefore, larger initial size patches can usually last longer and ultimately affect biogeochemical processes much stronger than smaller patches.

Micro RNA expression alterations in patients with glioblastoma

Glioblastoma, also known as glioblastoma multiform or GBM, is the most common and most malignant primary brain tumor. The clinical history of patients with glioblastoma is short, usually less than 3 months in more than 50% of cases after diagnosis. Currently, the methods of glioblastoma treatment are chemotherapy, radiotherapy and surgery. Even with the more effective treatment options, patients with glioblastoma most likely have a median survival time of 10 to 12 months. It is necessary to seek other treatment methods, including gene-targeted treatment. The success of gene-targeted treatment depends critically on the knowledge of genes that may be the cause of, or contribute to disease. To establish a correlate between glioblastoma survival timeline and micro RNA expression alteration, a study of 91 glioblastoma patients was conducted at the University of Texas M. D. Anderson Cancer Center. These 91 glioblastoma patients were newly diagnosed from 2002 to 2007. Statistical analysis was conducted to test the association of miRNA expression alteration between long-term survival and short-term survival glioblastoma. The completion of this proposed study will provide a better understanding of the regulatory role of miRNA in glioblastoma progression.^

LOW REACTIVE OXYGEN SPECIES AND HIGH GLYCOLYSIS IN GLIOBLASTOMA STEM CELLS:MECHANISMS AND THERAPEUTIC IMPLICATIONS

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of glioblastoma stem cells (GSCs), which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. Exposure of GSCs to fetal bovine serum (FBS) can cause their differentiation, the underlying mechanism of which remains unknown. Reactive oxygen species (ROS) play an important role in normal stem cell differentiation, but their role in affecting cancer stem cell fate remains unclear. Whether the metabolic characteristics of GSCs are different from other glioblastoma cells and can be targeted are also unknown. In this study, we used several stem-like glioblastoma cell lines derived from clinical tissues by typical neurosphere culture system or orthotopic xenografts, and showed that addition of fetal bovine serum to the medium induced an increase of ROS, leading to aberrant differentiation and decreases of stem cell markers such as CD133. We found that exposure of GSCs to serum induced their differentiation through activation of mitochondrial respiration, leading to an increase in superoxide (O2-) generation and a profound ROS stress response manifested by upregulation of oxidative stress response pathway. This increase in mitochondrial ROS led to a down-regulation of molecules including SOX2, and Olig2, and Notch1 that are important for stem cell function and an upregulation of mitochondrial superoxide dismutase SOD2 that converts O2- to H2O2. Neutralization of ROS by antioxidant N-acetyl-cysteine in the serum-treated GSCs suppressed the increase of superoxide and partially rescued the expression of SOX2, Olig2, and Notch1, and prevented the serum-induced differentiation phenotype. Additionally, GSCs showed high dependence on glycolysis for energy production. The combination of a glycolytic inhibitor 3-BrOP and a chemotherapeutic agent BCNU depleted cellular ATP and inhibited the repair of BCNU-induced DNA damage, achieving strikingly synergistic killing effects in drug resistant GSCs. This study uncovers the metabolic properties of glioblastoma stem cells and suggests that mitochondrial function and cellular redox status may profoundly affect the fates of glioblastoma stem cells via a ROS-mediated mechanism, and that the active glycolytic metabolism in cancer stem cells may provide a biochemical basis for developing novel therapeutic strategies to effectively eliminate GSCs.

Potential REST Targets in Neural Stem Cells and Glioblastoma-Derived Stem Cells

Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults. The current theory is that these tumors are caused by self-renewing glioblastoma-derived stem cells (GSCs). At the current time, the mechanisms that regulate self-renewal and other oncogenic properties of GSCs remain unknown. Recently, we found transcriptional repressor REST maintains self-renewal in neural stem cells (NSCs) and in GSCs. REST also regulates other oncogenic properties, such as apoptosis, invasion and proliferation. However, the mechanisms by which REST regulates these oncogenic properties are unknown. In an attempt to determine these mechanisms, we performed loss and gain-of-function experiments and genome-wide mRNA expression analysis in GSCs, and we were able to identify REST-regulated genes in GSCs. This was accomplished, after screening concordantly regulated genes in NSCs and GSCs, utilizing two RE1 databases, and setting two-fold expression as filters on the resulting genes. These results received further validation by qRT-PCR. Ingenuity Pathway Analysis (IPA) analysis further revealed the top REST target genes in GSCs were downstream targets of REST and/or involved in other cancers in other cell lines. IPA also revealed that many of the differentially-regulated genes identified in this study are involved in oncogenic properties seen in GBM, and which we believe are related to REST expression.

αvβ8 Integrin Interacts with RhoGDI1 to Regulate Rac1 and Cdc42 Activation and Drive Glioblastoma Cell Invasion

As an interface between the circulatory and central nervous systems, the neurovascular unit is vital to the development and survival of tumors. The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are major impediments to surgical resection and targeted therapies. Adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we have utilized human GBM cell lines, primary patient samples, and pre-clinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin associates with Rho GDP Dissociation Inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin-RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, suppresses activation of Rho proteins to promote GBM cell invasiveness. Hence, targeting the αvβ8 integrin-RhoGDI1 signaling axis may be an effective strategy for blocking GBM cell invasion.

The functional role of the tumor suppressor MMAC /PTEN in glioblastoma multiforme and prostate cancer

Investigations into the molecular basis of glioblastoma multiforme led to the identification of a putative tumor suppressor gene, MMAC/ PTEN. Initial studies implicated MMAC/PTEN in many different tumor types, and identified a protein phosphatase motif in its sequence. This project aimed to identify the biological and biochemical functions of MMAC/PTEN by transiently expressing the gene in cancer cells that lack a functional gene product. ^ Expression of MMAC/PTEN mildly suppressed the growth of U251 human glioma cells and abrogated the growth advantage mediated by overexpression of the epidermal growth factor receptor (EGFR). Immunoblotting demonstrated that MMAC/PTEN expression did not affect the phosphorylation of the EGFR itself, or the intermediates of several downstream signaling pathways. However, MMAC/PTEN expression significantly reduced the phosphorylation and catalytic activity of the proto-oncogene Akt/PKB. While Akt/PKB regulates the survival of many cell types, expression of MMAC/PTEN did not induce apoptosis in adherent U251 cells. Instead, MMAC/PTEN expression sensitized the cells to apoptosis when maintained in suspension (anoikis). As the survival of suspended cells is one of the hallmarks leading to metastasis, MMAC/PTEN expression was examined in a system in which metastasis is more clinically relevant, prostate cancer. ^ Expression of MMAC/PTEN in both LNCaP and PC3-P human prostate cancer cells specifically inhibited Akt/PKB phosphorylation. MMAC/PTEN expression in LNCaP cells resulted in a profound inhibition of growth that was significantly greater than that achieved with expression of p53. Expression of MMAC/PTEN in PC3-P cells resulted in greater growth inhibition than was observed in U251 glioma cells, but less than was observed in LNCaP cells, or upon p53 expression. To determine if MMAC/PTEN could function as a tumor suppressor in vivo, the effects of MMAC/PTEN expression on PC3-P cells implanted orthotopically in nude mice were examined. The ex-vivo expression of MMAC/PTEN did not decrease tumor incidence, but it did significantly decrease tumor size and metastasis. In-vivo expression of MMAC/PTEN in pre-established PC3-P tumors did not significantly inhibit tumor incidence or size, but did inhibit metastasis formation. ^ These studies demonstrate that MMAC/PTEN is a novel and important tumor suppressor gene, which functions to downregulate an important cell survival signaling pathway. ^

YKL -40, a vascular -associated serum marker for glioblastoma, correlates with chronic activation of AKT

YKL-40 is a secreted glycoprotein that has been reported to be expressed in pathologic conditions of extracellular matrix degradation and angiogenesis, such as rheumatoid arthritis, severe osteoarthritis, primary colorectal cancer, metastatic breast cancer, and recurrent ovarian cancer (Dehn, Hogdall et al. 2003). ^ We have identified YKL-40 as a serum marker for glioblastoma multiforme (GBM) using microarray analysis from samples of GBM. We compared the gene expression profile of 19 gliomas to pooled normal brain tissue using the Incyte 10,000 gene expression array. The most differentially expressed gene in this analysis was YKL-40; it was detected in GBM samples with a range of 3 to 62-fold elevation over normal brain. Western blot analysis of glioma samples for YKL-40 protein levels revealed substantial elevation in approximately 65% of GBMs, and undetectable levels in lower-grade gliomas and normal brain tissue. ELISA analysis on serum samples of glioma patients showed that YKL-40 levels were substantially elevated in many of the GBM patients. Statistical analysis indicated that in patients with glioma, serum YKL-40 levels correlate with tumor grade and potentially tumor burden in GBM. ^ Furthermore, we found that YKL-40 expression by in-situ hybridization on a brain tumor tissue array was limited to GBM's and gliosarcomas (GSA), and that YKL-40 expression was specific to the GBM component of GSA. Additional in-situ hybridization analysis, found it to be regionally associated with tumor vasculature as well as activated AKT expression in both human and mouse GBM's. Correlation of elevated YKL-40 with phospho-AKT was confirmed by Western blot analysis on a series of glioblastoma tumors, and inhibition of PI3 Kinase signaling by addition of LY294002 also decreased secretion of YKL-40 over a 7-day period in U87 glioblastoma cell tine. Lastly, YKL-40 expression was induced in response to serum starvation and altered by interaction with specific extracellular matrix (ECM) modules. In summary, we have identified the first accurate serum marker for high-grade gliomas. Furthermore, our findings indicate that YKL-40 is a highly expressed vascular-related glycoprotein in human GBM tissue and that it is affected by the AKT signaling pathway and interaction with components of brain ECM proteins. ^